The inaccessibility of living bone marrow hampers the study of its pathophysiology under myelotoxic stress induced by drugs, radiation or genetic mutations. Here, we show that a vascularized human bone-marrow-on-a-chip supports the differentiation and maturation of multiple blood-cell lineages over 4 weeks while improving CD34+ cell maintenance, and that it recapitulates aspects of marrow injury, including myeloerythroid toxicity after clinically relevant exposures to chemotherapeutic drugs and ionizing radiation as well as marrow recovery after drug-induced myelosuppression. The chip comprises a fluidic channel filled with a fibrin gel in which CD34 + cells and bone-marrow-derived stromal cells are co-cultured, a parallel channel lined by human vascular endothelium and perfused with culture medium, and a porous membrane separating the two channels. We also show that bone-marrow chips containing cells from patients with the rare genetic disorder Shwachman–Diamond syndrome reproduced key haematopoietic defects and led to the discovery of a neutrophil-maturation abnormality. As an in vitro model of haematopoietic dysfunction, the bone-marrow-on-a-chip may serve as a human-specific alternative to animal testing for the study of bone-marrow pathophysiology.

Recent advances in single-cell transcriptomics techniques have opened the door to the study of gene regulatory networks (GRNs) at the single-cell level. Here, we studied the GRNs controlling the emergence of hematopoietic stem and progenitor cells from mouse embryonic endothelium using a combination of single-cell transcriptome assays. We found that a heptad of transcription factors (Runx1, Gata2, Tal1, Fli1, Lyl1, Erg and Lmo2) is specifically co-expressed in an intermediate population expressing both endothelial and hematopoietic markers. Within the heptad, we identified two sets of factors of opposing functions: one (Erg/Fli1) promoting the endothelial cell fate, the other (Runx1/Gata2) promoting the hematopoietic fate. Surprisingly, our data suggest that even though Fli1 initially supports the endothelial cell fate, it acquires a pro-hematopoietic role when co-expressed with Runx1. This work demonstrates the power of single-cell RNA-sequencing for characterizing complex transcription factor dynamics.

The endothelial to haematopoietic transition (EHT) is the process whereby haemogenic endothelium differentiates into haematopoietic stem and progenitor cells (HSPCs). The intermediary steps of this process are unclear, in particular the identity of endothelial cells that give rise to HSPCs is unknown. Using single-cell transcriptome analysis and antibody screening, we identify CD44 as a marker of EHT enabling us to isolate robustly the different stages of EHT in the aorta-gonad-mesonephros (AGM) region. This allows us to provide a detailed phenotypical and transcriptional profile of CD44-positive arterial endothelial cells from which HSPCs emerge. They are characterized with high expression of genes related to Notch signalling, TGFbeta/BMP antagonists, a downregulation of genes related to glycolysis and the TCA cycle, and a lower rate of cell cycle. Moreover, we demonstrate that by inhibiting the interaction between CD44 and its ligand hyaluronan, we can block EHT, identifying an additional regulator of HSPC development.

Highlights d Injury-exposed satellite cells (SCs) rapidly and transiently induce FOS d FOS-expressing SCs activate faster and display superior regenerative activity d Fos-null SCs activate more slowly and have diminished regenerative activity d The FOS target Art1 is required for efficient SC activation and muscle repair

GCY. CEJ was supported by NIH training grant T32 CA070083, a postdoctoral fellowship F32 HL124941, and a Claudia Adams Barr Award from the Dana-Farber Cancer Institute. We thank Formation Biologics for supplying AVID200. We thank Donna Neuberg for critical scientific advice. We thank the Dana-Farber Cancer Institute Flow Cytometry Core, especially John Daley, Michael Buonopane, and Alexander Heubeck, for providing technical expertise and equipment; Broad Technology Labs, especially Jim Bochicchio and Caroline Cusick, for providing project management support; the Beth Israel Deaconess Medical Center Genomics, Proteomics, Bioinformatics and Systems Biology Center, especially Simon T. Dillon, Xuesong Gu, and Hasan Out, for providing technical and analytical support; and the Broad Genomics Platform.

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